The ability of cells to receive and interpret cues directing them to express genes appropriate for their location is central to the process of embryonic development. Most cells of the embryo must respond to cues in both dorso-ventral (DV) and anterior-posterior (AP) axes. We have developed a unique set of reagents that make analysis of expression from the Pax3 gene in the mouse a particularly tractable system for understanding how DV and AP positional information is integrated at the level of a single gene in establishing the central body plan of mammals. Pax3 is an effector gene which functions in translating DV positional information into differentiation of discrete cell lineages within the neural tube and somites. Its initial expression domain marks what will ultimately be the dorsal pole of the embryo. Four specific aims will be accomplished towards understanding the mechanisms that restrict Pax3 expression to this domain. First, in the mouse, we hypothesize that Bmp4 inhibits Pax3 expression in a posterior-ventral domain and integration of this inhibitory signal with stimulatory signals, required for dorsal localization, establish the appropriate pattern of expression. This role for Bmp4 is different from that in the chick and implies a difference in the fundamental process of DV patterning between these two vertebrates. These results will be carefully examined using both embryo culture and transgenic analysis. Aims 2 and 3 will define the molecular basis by which Bmp4 signals are interpreted on the Pax3 promoter, and integrated with other signaling inputs, at the level of cis-acting DNA sequence elements. Our previous work has localized sequence elements required for Pax3 expression in the neural tube to 1.6 kbp of DNA sequence, and we have now refined the required elements to four discrete sites. The roles of these elements in vivo will be determined. Finally, proteins that bind the elements sequence specifically have been isolated. Aim 4 will identify these proteins and generate cDNAs and Abs to allow us to define their functional roles. These studies will move our understanding of how DV pattern is established, and overlaid by AP patterning mechanisms, to localize Pax3 expression in mammals to a new level of resolution.